EP1124979A4 - Production d'acide 3-hydroxypropionique chez des organismes de recombinaison - Google Patents

Production d'acide 3-hydroxypropionique chez des organismes de recombinaison

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Publication number
EP1124979A4
EP1124979A4 EP00959652A EP00959652A EP1124979A4 EP 1124979 A4 EP1124979 A4 EP 1124979A4 EP 00959652 A EP00959652 A EP 00959652A EP 00959652 A EP00959652 A EP 00959652A EP 1124979 A4 EP1124979 A4 EP 1124979A4
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Prior art keywords
glycerol
hydroxypropionic acid
aldehyde dehydrogenase
seq
gene
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German (de)
English (en)
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EP1124979A1 (fr
EP1124979B1 (fr
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Patrick F Suthers
Douglas C Cameron
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/0103—Glycerol dehydratase (4.2.1.30)

Definitions

  • Native microorganisms have the ability to produce 1 ,3 - propanediol from glycerol as well.
  • Commercial interests are exploring the production of 1,3 - propanediol from glycerol or glucose, in recombinant organisms which have been engineered to express the enzymes necessary for 1,3 - propanediol production from other organisms.
  • 3 - hydroxypropionic acid CAS registry Number [503-66-2] (abbreviated as 3- HP) is a three carbon non-chiral organic molecule.
  • the IUPAC nomenclature name for this molecule is propionic acid 3 - hydroxy. It is also known as 3 - hydroxypropionate, ⁇ - hydroxpropionic acid, ⁇ - hydroxypropionate, 3 - hydroxypropionic acid, 3 - hydroxypropanoate, hydracrylic acid, ethylene lactic acid, ⁇ -lactic acid and 2 - deoxyglyceric acid.
  • 3-HP for commercial use is now commonly produced by organic chemical syntheses.
  • the 3-HP produced and sold by these methods is relatively expensive, and it would be cost prohibitive to use it for the production of monomers for polymer production.
  • some organisms are known to produce 3-HP. However, there is not yet available a catalog of genes from these organisms and thus the ability to synthesize 3-HP using the enzymes natively responsible for the synthesis of that molecule in the native hosts which produce it does not now exist.
  • PHB Poly(3 - hydroxybutyrate)
  • PHAs polyhydroxyalkonates
  • Ralstonia eutropha (“Alcaligenes eutrophus") and Pseudomonas oleovorans. Both Ralstonia eutropha and Pseudomonas oleovorans are able to grow on a nitrogen free media containing 3 - hydroxy - propionic acid, 1,5 - pentanediol or 1,7 - heptanediol.
  • poly(3 - hydroxybutyrate - co - 3 - hydroxypropionic acid) is formed containing 7 mol % 3 - hydroxypropionic acid.
  • These cells also store 3 mol %, 3 - hydroxypropionic acid poly(3 - butyrate - co - 3 - hydroxypropionic acid).
  • An E. coli strain engineered to express PHA synthase from either Ralstonia eutropha or Zoolgoea ramigera produced poly(3 - hydroxypropionic acid) when feed 1,3 - propanediol.
  • Glycerol dehydratase found in the bacterial pathway for the conversion of glycerol to 1,3 - propanediol, catalyzes the conversion of glycerol to 3 - hydroxypropionaldehyde and water.
  • This enzyme has been found in a number of bacteria including strains of Citrobacter, Klebsiella, Lactobacillus, Entrobacter and Clostridium.
  • 1,3 - propanediol pathway a second enzyme 1,3 - propanediol oxido-reductase (EC 1.1.202) reduces 3 - hydroxypropanaldehyde to 1,3 - propanediol in a NADH dependant reaction.
  • Aldehyde dehydrogenases are enzymes that catalyze the oxidation of aldehydes to carboxylic acids.
  • the genes encoding non-specific aldehyde dehydrogenases have been identified in a wide variety of organisms e.g.; ALDH2 from Homo sapiens, ALD4 from Saccharomyces cerevisiae, and from E. coli both aldA and aldB, to name a few. These enzymes are classified by co-factor usage, most require either AND + , or NADP + and some will use either co-factor.
  • the genes singled out for mention here are able to act on a number of different aldehydes and it likely that they may be able to oxidize 3 - hydroxy - propionaldehyde to 3 - hydroxypropionic acid.
  • the present invention is intended to permit the creation of a recombinant microbial host which is capable of synthesizing 3-HP from a starting material of glycerol or glucose.
  • the glycerol or glucose is converted to 3 - hydroxypropionicaldehyde (abbreviated as 3-HP A) which is then converted to 3-HP.
  • This process requires the so-called dhaB gene from Klebsiella pneumoniae which encodes the enzyme glycerol dehydratase any one of four different aldehyde dehydrogenase genes to convert 3-HPA to 3-HP.
  • the four aldehyde dehydrogenase genes used were aldA from the bacterium E. coli, ALDH2 from humans, ALD4 from the yeast Saccharomyces cerevisiae, and aldB from E. coli. The yeast gene appeared to give the best results.
  • a difficulty in the realization of the production of 3-HP desired here is that ribosome binding sites from non-native hosts are often ineffectual and lead to poor protein production and that many non-native promoters are often poorly transcribed and a bar to high protein expression.
  • the inventors also recognized that a non- native promoter that is known to be very active and is inducible by the addition of a small molecule unrelated to the pathway being expressed is often a very efficient way to express and regulate the levels of enzymes expressed in hosts such as E. coli.
  • To achieve high levels of regulated gene expression plasmids were constructed which placed the expression of all exogenous genes necessary for the production of 3 - hydroxypropionic acid from glycerol under the regulation of the trc promoter.
  • the trc promoter is efficient, not native to E. coli, and inducible by the addition of IPTG.
  • the present specification describes a genetic construct for use in the production of 3 - hydroxypropionic acid from glycerol.
  • the genetic construct includes exemplary DNA sequences coding for the expression of a glycerol dehydratase and a DNA sequence coding for aldehyde dehydrogenase.
  • the set of exemplary sequences necessary for the expression of glycerol dehydratase is collectively referred to as "dhaB" .
  • the set of sequences necessary for the expression of aldehyde dehydrogenase includes any one of four different genes which proved efficacious.
  • the enzymes necessary for the production of 3 - hydroxypropionic acid from glycerol in E. coli were expressed under the regulation of the trc promoter, a non-native promoter inducible by the addition of IPTG.
  • the glycerol dehydratase was encoded by the dhaB gene from Klebsiella pneumoniae, the aldehyde dehydrogenases used was any one of four different genes (ALDH2 from Homo sapiens, ALD4 from S. cerevisiae, aldB from E. coli or aldA from E. coli).
  • the only genetic elements present that would be necessary are the structural genes dhaB and an aldehyde dehydrogenase gene encoding a protein that efficiently catalyzes the oxidation of 3-hydroxypropionaldehyde to 3 -hydroxypropionic acid, and non-native promoter sequences specifically selected to give the type of inducible control most appropriate for the context of the process in which the construct is to be used. Extraneous pieces of DNA, whether retained in the construct or added from other DNA sequences, would not necessarily be detrimental to effective 3-HP synthesis by the host organism, but would not be needed.
  • Each sequence to be translated would necessarily be preceded by a ribosome binding site, functional in the selected host so that the messenger RNA(s) coding for the proteins of interest could be translated by ribosomes. Terminator sequences immediately downstream of each translated unit would also be necessary in some organisms, particularly in eukaryotes.
  • the construct could be part of an autonomously replicating sequence, such as a plasmid or phage vector, or could be integrated into the genome of the host.
  • the structural genes and appropriate promoters could be isolated by the use of restriction enzymes, by the polymerase chain reaction (PCR), by chemical synthesis of the appropriate oligonucleotides, or by other methods apparent to those skilled in the art or molecular biology.
  • the promoter(s) would be derived from genomic DNA of other organism or from artificial genetic constructs containing promoters. Appropriate promoter fragments would be ligated into the construct upstream of the structural genes in any one of several possible arrangements.
  • the aldehyde dehydrogenase expressed would have: high specific activity towards 3-hydroxypropionaldehyde; be very stable in the host it is expressed in; be readily over expressed in the selected host; not be inhibited by either the substrates necessary for the reaction or the products formed by the reaction; be fully active under the fermentation conditions most favorable for the production of 3 - hydroxypropionic acid and be able to use either NAD + or NADP + .
  • One possible arrangement is the true operon, where one promoter is used to direct transcription in one direction of all necessary Open Reading Frames (ORFs). The entire message is then contained in one messenger R A.
  • the advantages of the operon are that it is relatively easy to construct, since only one promoter is needed; that is it is relatively simple to replace the promoter with another promoter if that would be desirable later; and that it assures that the two genes are under the same regulation.
  • the main disadvantage of the operon scheme is that the levels of the expression of the two genes cannot be varied independently. If it is found that the genes, for optimal 3 - hydroxypropionic acid synthesis, should be expressed at different levels, the operon in most cases cannot be used to realize this.
  • Another possible arrangement is the multiple-promoter scheme. Two or more promoters, with the same or distinct regulatory behavior, could be used to direct transcription of the genes.
  • one promoter could be used to direct transcription of dhaB and one to direct transcription of the gene encoding the appropriate aldehyde dehydrogenases. Because the genes theoretically can be transcribed and translated separately, a great number of combinations of multiple promoters is possible. Additionally, it would be most desirable to prevent the promoters from interfering with one another. This could be achieved either by placing two promoters into the construct such that they direct transcription in opposite directions, or by inserting transcriptional terminator sequences downstream of each separately transcribed unit.
  • the main advantage of the multiple-promoter construct is that it permits independent regulation of as many distinct units as desired, which could be important.
  • the promoter sequence(s) used should be functional in the selected host organism and preferably provide sufficient transcription of the genes comprising the glycerol to 3 - hydroxypropionic acid pathway to enable the construct to be adequately active in that host.
  • the promoter sequence(s) used would also effect regulation of transcription of the genes enabling the glycerol to 3-HP pathway to be adequately active under the fermentation conditions employed for 3-HP production, and preferably they would be inducible, such that expression of the genes could be modulated by the inclusion in, or exclusion from, the fermentation of a certain agents or conditions.
  • one promoter which induced by the addition of an inexpensive chemical (the inducer) to the medium, could control transcription of both the dhaB gene and the gene encoding the appropriate aldehyde dehydrogenase.
  • the cells would be permitted to grow in the absence of the inducer until they accumulated to a predetermined level.
  • the inducer would then be added to the fermentation and nutritional changes commensurate with the altered metabolism would be made to the medium as well.
  • the cells would then be permitted to utilize the substrate(s) provided for 3-HP production (and additional biomass production if desired). After the cells could no longer use substrate to produce 3-HP, the fermentation would be stopped and the 3-HP recovered.
  • glycerol dehydratase and a suitable aldehyde dehydrogenase the two enzymes necessary for the production of 3 - hydroxypropionic acid from glycerol, it is required that the DNA sequences containing the glycerol dehydratase and aldehyde dehydrogenase coding sequences be combined with at least a promoter sequence (preferably a non-native promoter although some native promoter activity may be present).
  • a promoter sequence preferably a non-native promoter although some native promoter activity may be present.
  • Sequences 1, 3, 5 and 7 present different native genomic sequences for genes encoding aldehyde dehydrogenases.
  • SEQ ID NO: 1 contains the full native DNA sequence encoding the ALD4 enzyme from Saccharomyces cerevisiae.
  • the amino acid sequence of the protein is presented as SEQ ID NO:2.
  • SEQ ID NO:3 includes the DNA sequence for the human ALDH2 gene, again including the full protein coding region.
  • the amino acid sequence for this human alcohol dehydrogenase is presented in SEQ ID NO:4.
  • SEQ ID NO:5 and 7 respectively present the full coding sequences from the E. coli genes aldA and aldB, both of which encode alcohol dehydrogenases.
  • the amino acid sequences for the proteins encoded by the genes are presented in SEQ ID NO: 6 and 8 respectively.
  • SEQ ID NO:9 contains the native genomic DNA sequence for the dhaB gene from the dha regulon of Klebisiella pneumoniae.
  • the coding sequences for this complex regulon produces five polypeptides, which are presented as SEQ ID NOS: 10 through 13, which together provide the activity of the glycerol dehydratase enzyme.
  • Each of these coding sequences can be used to make genetic constructs for the expression of the appropriate enzymes in a heterologous hosts.
  • heterologous promoters will be joined to the coding sequences for the enzymes, but all that it required is that the promoters be effective for the hosts in which the genes are to be expressed. It is also contemplated and envisioned that significant variations in DNA sequence are possible from the native DNA coding sequences presented here. As is well known in the art, due to the degeneracy of the genetic code, many different DNA sequences can encode the expression of the same protein. So, when this document uses language specifying a DNA sequence encoding a protein, it is intended to encompass any DNA sequence which can be used to express that protein even if different from the genomic sequences presented here.
  • the primers aldA_L (SEQ ID NO: 14), and aldA_R (SEQ ID NO: 15), were used to amplify the 1513 bp aldA fragment from genomic E. coli DNA (strain MG1655, a gift from the Genetic Stock Center, New Haven, CT).
  • the gel purified PCR fragment containing a DNA sequence coding for the expression of aldehyde dehydrogenase was inserted into Ncol-Xhol site of pSE380 (Invitrogen, San Diego, CA) to give pPFS3.
  • the resulting plasmid contained aldA under the control of the trc promoter. This construct allowed for high-level expression of the aldA gene from E. coli under regulation of the trc promoter. Unless indicated otherwise all molecular biology and plasmid constructions were done in E. coli AG1 (Stratagene, La Jolla, CA).
  • the resulting PCR converted the TGA stop codon into a TAA stop codon.
  • the gel- purified PCR fragment containing the DNA sequence sufficiently coding for the expression of aldehyde dehydrogenase was inserted into the Kpnl-Sacl site of pSE380 to give pPFS12.
  • the primers ALD4_L (SEQ ID NO : 16), and ALD4_R (SEQ ID NO : 17), were used to amplify the 1595 bp ALD4 fragment from S. cerevisiae DNA (strain YPH500).
  • the gel-purified fragment containing a DNA sequence coding for the expression of aldehyde dehydrogenase was inserted into the Kpnl-Sacl site of pPFS3 to give pPFS8.
  • the resulting plasmid contained mature ALD4 under control of the trc promoter.
  • the primers ALDH2_L (SEQ ID NO:18), and ALDH2_R (SEQ ID NO:19), were used to amplify the 1541 bp ALDH2 fragment from pT7-7::ALDH2, a gift from H. Weiner (Purdue University, West Lafayette, IN).
  • the gel purified PCR fragment containing a DNA sequence sufficiently homologous to base pairs 22 to 1524, inclusive of SEQ ID NO : 3 so as to code for the expression of aldehyde dehydrogenase was inserted in to the Kpnl-Sacl site of pSE380 to give pPFS7.
  • This sequence was moved from pPFS7 into the Kpnl-Sacl site of pPFS3 to give pPFS9.
  • the resulting plasmid contained mature ALDH2 under the control of the trc promoter.
  • the primers pTRC_L (SEQ ID NO:22), and pTRC_R )SEQ ID NO:23), were used to amplify the 540 bp fragment from pSE380.
  • the gel purified PCR fragment was inserted into the Hpal-Kpnl site of pPFS3 to give pPFS13.
  • the resulting plasmid deleted the "native" ribosome binding site of pSE380 and a Ncol site (which contained an extraneous ATG start codon upstream of the cloned genes).
  • the Kpnl-Sacl fragments of pPFS8, pPFS9, and pPSF12 were inserted into the Kpnl-Sacl site of pPFS13 to give pPFS14, pPFS15, and pPFSl ⁇ , respectively.
  • fermentation products can be quantified with a Waters Alliance Integrity HPLC system (Milford, MA) equipped with a refractive index detector, a photodiode array detector, and an Aminex HPX-87H (Bio- Rad, Hercules, CA) organic acids column.
  • the mobile phase should be 0.01 N sulfuric acid solution (pH 2.0) at a flow rate of 0.5 rnL/min.
  • the column temperature should be set to 40 °C.
  • Compounds can be identified by determining if they co-elute with authentic standards. Prior to analysis, all samples should be filtered through 0.45 ⁇ M pore size membrane.
  • Aldehyde dehydrogenase activity can be determined by measuring the reduction of ⁇ -NAD + at 25 °C with 3 - hydroxypropionaldehyde as a substrate.
  • All buffers should contain 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM Pefabloc SC (Boehringer Mannheim, Indianapolis, IN) and 1 mM Tris (carboxyethyl) phosphine hydrochloride (TCEP-HCL).
  • EDTA ethylenediaminetetraacetic acid
  • Pefabloc SC Boehringer Mannheim, Indianapolis, IN
  • Tris (carboxyethyl) phosphine hydrochloride TCEP-HCL
  • the solution should contain 100 mM Tris HCL Buffer (pH 8.0), 100 mM KC1.
  • ALDH2 the solution should contained 100 mM sodium pyrophosphate (pH 9.0).
  • the solution should contain 20 mM sodium glycine (pH 9.5). A total of 3.0 mL of buffer should be added to quartz cuvettes and allowed to equilibrate to assay temperature. From 5 to 20 ⁇ L of cell extract should be added and background activity recorded after the addition of ⁇ -NAD + to a final concentration of 0.67 mM. The reaction should be started by the addition of substrate (either acetaldehyde, propionaldehyde, or 3 - hydroxypropionaldehyde) to a final concentration of 2 mM. Assay mixtures should be stirred with micro-stirrers during the assays.
  • substrate either acetaldehyde, propionaldehyde, or 3 - hydroxypropionaldehyde
  • aldehyde dehydrogenase activity assays one unit is defined as the reduction of 1.0 ⁇ M of ⁇ -AND + per minute at 25 ° C. These reactions can be monitored by following the change in absorbence at 340 nm (A 340 ) at 25 °C on a Varain Carry- 1 Bio spectrophotometer (Sugar Land, TX). Total protein concentrations in the cell extracts can be determined using the Bradford assay method (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.
  • Klebsiella pneumoniae expresses glycerol dehydratase, an enzyme that catalyzes the conversion of glycerol to 3 - hydroxypropionaldehyde, (dhaB) and 1,3 - propanediol oxidoreductase an enzyme that catalyzes the conversion of 3 - hydroxypropionaldehyde to 1 ,3 - propanediol respectively (the gene product from dhaT).
  • a plasmid encoding these two genes was created and expressed in E. coli (plasmid pTC53). The dhaT gene was deleted from pTC53 to create pMH34.
  • the resulting plasmid still contained the DNA sequence complementary to base pairs 330 to 2153 inclusion of SEQ ID NO : 9, the complement of base pairs 2166 to 2591 , inclusive, of SEQ ID NO : 9, and the complement of base pairs 3191 to 4858, inclusive, of SEQ ID NO : 9, so as to code for the expression of glycerol dehydratase.
  • the fragment of DNA encoding these sequences was excised from pMH34 by cutting it with Sall-Xbal, and the resulting fragment was gel purified (the purified fragment was gift from M. Hoffman ofthe University of Wisconsin - Madison). This DNA fragment was inserted into the Satl-Xbal site of pPFS13 to give pPFS17.
  • the resulting plasmid contained both the aldA and dhaB genes under the control ofthe trc promoter. Similarity, the gel-purified Sall-Xbal fragment from pMH34 was inserted into the Sall-Xbal sites of pPFS14, pPFS15, and pPFSl ⁇ to give pPFS18, pPFS 19, and pPFS20, respectively. These plasmids contained ALD4, ALDH2, and aldB, respectively, as well as dhaB under the control ofthe trc promoter; in all ofthe constructs the dhaB gene were downstream ofthe gene encoding the aldehyde dehydrogenase. Expression in E. coli.
  • the efficacy of E. coli as a platform for the production of 3-HP from growth on glucose has been examined using a mathematical model developed for this purpose.
  • the model was executed in two different ways assuming the conversion of one mole of glucose under either anaerobic or aerobic conditions either directly to 3-HP or to the production of 3-HP and ATP.
  • the optimum yield under anaerobic conditions is 1 mole of 3-HP and 1 mole of lactate.
  • the more realistic yield under anaerobic conditions is 0.5 moles of 3-HP, 1.5 moles of lactate and 1 mole of ATP.
  • the optimum yield under aerobic conditions is 1.9 moles of 3-HP and 0.3 moles of CO 2 .
  • the realistic yield under aerobic conditions is 1.85 moles of 3-HP, 0.35 moles of CO 2 and 1 mole of ATP.
  • the effect of 3-HP concentration on E. coli strain MG1655 growth was measured. Cells were grown on standard media with and without the addition of up to 80g/L of 3-HP. The best fit of these data demonstrated that 3-HP was only 1.4 times as inhibitory as lactic acid on the growth of E. coli. It is possible to economically produce lactic acid using E. coli, since 3-HP is only 1.4 times more inhibitory than lactic acid, it should be possible to use E. coli as a host for the commercial production of 3-HP.
  • the standard media contained the following per liter: 6 g Na 2 HPO 4 , 3 g KH 2 PO , 1 g NH 4 C1, 0.5 g NaCl, 3 mg CaCl 2 , 5 g yeast extract (Difco Laboratories, Detroit, MI) and 2 mM MgSO 4 .
  • ampicillin 100 mg/mL was added to the media.
  • Isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added in varying amounts to induce gene expression. All fermentations were carried out in an incubator- shaker at 37 C and 200 rpm. Anaerobic fermentations were carried out in 500-mL anaerobic flasks with 300 mL of working volume.
  • Inocula for fermentations were grown overnight in Luria-Bertani medium supplemented with ampicillin is necessary.
  • the 300-mL fermentations were inoculated with 1.5 mL ofthe overnight culture.
  • enzyme assays fermentations were incubated for 24 hours.
  • Cells were harvested by centrifugation at 3000 x g for 10 minutes at 4°C with a Beckman (Fullerton, CA) model J2-21 centrifuge. Cell pellets were washed twice in 100 mM potassium phosphate buffer at pH 7.2 and re-suspended in appropriate assay resuspension buffer equal to 5 x ofthe volume ofthe wet cell mass. The cells were homogenized using a French pressure cell. The homogenate was centrifuged at 40000 x g for 30 minutes. The supernatant was dialyzed against the appropriate resuspension buffer using 10000 molecular weight cut-off pleated dialysis tubing (Pierce, Rockford, IL) at 4°C. Dialysis buffer was changed after 2 hours, and 4 hours, and dialysis was stopped after being allowed to proceed overnight.
  • E. coli AG1 cells transfected with the plasmids constructed to express the aldA, ALD4, ALDH2, or aldB genes were grown in 500-mL anaerobic flasks. Twelve hours after the fermentations were inoculated IPTG was added to induce enzyme expression. The cells were allowed to grow for an additional 12 hours then harvested and lysed as discussed above.
  • the soluble fraction ofthe lysate was assayed for aldehyde dehydrogenase activity using the substrate 3 -hydroxy propionicaldehyde in the buffer appropriate for the particular enzyme expressed
  • the plasmid, aldehyde dehydrogenase expressed and specific activity measured were as follows: pPFS13, aldA, 0.2; ⁇ S14, ALD4, 0.5, pPFSl 5, ALDH2, 0.3; and pPFSl ⁇ , aldB. 0.1.
  • the control E. coli strain AG1 harboring plasmid pSE380, encoded no exogenous aldehyde dehydrogenase activity and it had no detectable activity with 3-HP as substrate.
  • the cells were grown on standard media supplemented with 6 ⁇ M of Coenzyme B 12 , under anaerobic conditions in the absence of light (to protect the integrity ofthe Coenzyme B 12 necessary for DhaB activity). After 12 hours, IPTG was added to induce expression ofthe genes under the trc promoter at the same time 5 g L of glycerol was added.
  • the fermentation broth was assayed for 3 - HP by HPLC and GC, the plasmid, aldehyde dehydrogenase gene expressed and g/L of 3- HP measured were as follows: pSF17, aldA, 0.031; pPSF18 ALD4, 0.173; and pPSF19, ALDH2, 0.061.
  • Cells expressing dhaB but no exogenous aldehyde dehydrogenase genes (plasmid pMH34) produced 0.015 g/L of 3 - HP.
  • Cells expressing aldA, ALD4, ALDH2 or aldB but not dhaB (plasmids pPFS 13 , pPFS 14, pPFS 15 , pPFS 16, respectively) all produced less then 0.005 g/L of 3-HP when the media the cells were growing in was supplemented with 2.5g/L of 3 -hydroxypropionaldehyde.
  • vector pNH2 for example, to express the 3 -hydroxypropionic acid pathway in Rhodobacter capsulatus, one could obtain vector pNH2 from the American Type Culture Collection ( ATTC). This is a shuttle vector for use in R. capsulatus and E. coli. Organisms such as Saccharomyces cerevisiae which can convert glucose to glycerol could be used as a host, such a construct would enable the production of 3 - HP directly from glucose. Additionally, other substrates such as xylan could also be used given the selection of an appropriate host. Stochiometric analysis shows that best stochiometric yield of 3-HP production in
  • E. coli calculated on the basis of glucose consumed is obtained under aerobic conditions.
  • CO 2 is the only carbon-containing co-product, in particular the generation of lactic acid which occurs under anaerobic conditions is avoided.
  • Production of 3-HP under these conditions could result in a more economical recovery of 3-HP from the fermentation broth.
  • the dhaB gene and a gene encoding the appropriate aldehyde dehydrogenase could be cloned into the multiple cloning site of this vector in E. coli to facilitate construction, and then transformed into R. capsulatus.
  • the R. capsulatus nifH promoter, provided on the plasmid could be used to direct the transcription in R. capsulatus ofthe genes placed into pNF2 in series with one promoter, or with two copies ofthe nifH promoter. Expression ofthe genes in other organisms would require a procedure analogous to that presented here.
  • a program of directed evolution could be undertaken to select for suitable aldehyde dehydrogenases or they could be recovered from native sources, the genes encoding these enzymes in conjunction with a gene encoding an appropriate glycerol dehydratase activity, would then be made part of any ofthe constructs envisioned here to produce 3 - hydroxypropionic acid from glycerol.
  • a similar program of enzyme improvement including for example directed evolution could be carried out using the dhaB gene from Klebsiella pneumoniae as a starting point to obtain other variants of glycerol dehydratase that are superior in efficiency and stability to the form used in this invention.
  • enzymes which catalyzes the same reaction may be isolated from others organisms and used in place of the Klebsiella pneumoniae glycerol dehydratase. Such enzymes may be especially useful in alternative hosts wherein they may be more readily expressed, be more stable and more efficient under the fermentation conditions best suited to the growth ofthe construct and the production and recovery of 3-HP.

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EP00959652A 1999-08-30 2000-08-30 Production d'acide 3-hydroxypropionique par des organismes recombinants Expired - Lifetime EP1124979B1 (fr)

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EP1124979A1 (fr) 2001-08-22
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ATE338139T1 (de) 2006-09-15
BR0008355A (pt) 2002-07-16
AU7093600A (en) 2001-03-26

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